Project Summary/Abstract: Cystic Fibrosis (CF) is a genetic disorder that affects the major airways of the lungs of more than 30,000 people in the United States. The altered airways of 80% of CF patients are afflicted with a persistent bacterial colonization by Pseudomonas aeruginosa. This pathogen is 1 of the terminal pathogens isolated from the CF lung and is highly associated with extensive pulmonary damage. P. aeruginosa is extremely difficult to eradicate in the CF lung using conventional antibiotic therapies. This results in recurrent bacterial infections of the lower respiratory tract, which are the primary cause of morbidity and mortality. Several lines of evidence indicate that colonization of the CF lung by P. aeruginosa involves the biofilm mode of growth. Most significantly, is that biofilms are up to 1000-fold more resistant to antibiotics and biocides than are planktonic bacteria. The biofilm mode of growth has not been considered in antibiotic drug discovery and development. In preliminary work, we have identified potential targets that are required for regulating biofilm formation. These novel targets are proteins containing GGDEF (diguanylate cyclase) and EAL (phosphodiesterase) motifs, which are involved in signal transduction pathways that utilize the secondary messenger cyclic-diGMP. These proteins represent a class of novel drug targets with biochemical activities that are amenable to high-throughput screening assays. Our overall goal is to discover and develop drugs that inhibit the biofilm mode of growth in P. aeruginosa. Our strategy for reaching this goal is to identify GGDEF/EAL motif-containing proteins essential for biofilm formation, validate them as targets for anti-biofilm drug discovery, and identify small molecules that inhibit them and disrupt biofilm formation and/or maintenance in P. aeruginosa. In this proposal, we will achieve the following specific aims: (1) Identify GGDEF/EAL mutants that affect the regulation of biofilm formation in P. aeruginosa PA01. (2) Verify that the activities of GGDEF/EAL proteins required for modulating c-diGMP levels are essential for specifically regulating biofilm formation in several isolates of P. aeruginosa. (3) Identify additional components of the c-diGMP-dependent signaling pathway(s) and their effectors that are involved in regulating biofilm formation for verified GGDEF/EAL biofilm targets. Project Narrative: The development of drugs that block the most prevalent CF pathogens from adopting the biofilm mode of growth and developing intrinsic antibiotic resistance mechanisms will represent a significant advance in the treatment of CF. This will enable clinicians to prevent or to eradicate persistent infections in Cystic Fibrosis patients using a combination of these biofilm-blocking drugs and an antibiotic that is in clinical use. This development will represent a major step forward in the treatment of CF lung disease, and will have a profound economic and social impact; enabling clinicians to better control the biofilm-related infections that plague CF patients. [unreadable] [unreadable] [unreadable]